This proposal will help to understand the role of the antioxidant protein peroxiredoxin 1 (Prdx1) in natural killer cell function and tumor susceptibility. Reactive oxygen species (ROS) are one cause of aging and many diseases such as atherosclerosis, autoimmune disorders, neuronal degeneration and cancer. One role of antioxidants is to balance the redox homeostasis to avoid damaging excessive levels of ROS. Prdx1 is also known to be a natural killer (NK) cell activator from red blood cells (RBCs). Loss of Prdx1 in mice leads to premature death due to hemolytic anemia and cancer. Prdx1 deficient mice show decreased NK cell activity and increased cellular damage such as protein oxidation and DNA damage in murine embryonic fibroblasts (MEFs). The Prdx1 deficient mice provide an excellent tool to study the impact of ROS in disease development. Aim 1. To define the role of Prdx1 in natural killer cell activation. It is not fully understood how exogenous Prdx1 activates NK cells. Therefore, it will be demonstrated that Prdx1 is probably secreted by RBCs and co-localizes with the NK cell membrane or cytosol. Localization studies will be done by using fluorescence activated cell sorter (FACS) analysis, immunofluorescence or subcellular fractionation. NK cells will be examined for their ROS content, pH and calcium (Ca2+) content by using fluorescence dyes. Signaling of NK cells will be studied by Western blotting. RBCs and NK cells need Prdx1 to express Tumor Necrosis Factor alpha, I will study whether this production is dependent on Prdx1 antioxidant activity. All studies will include RBCs and NK cells from Prdx1 wildtype and null mice and recombinant Prdx1. Aim 2 will investigate Prdx1 and oxidative stressors as specific regulators of cellular transformation. It is not fully understood whether ROS play a specific role in cellular transformation. MEFs from Prdx1 wildtype and null mice will be chronically stressed with H202 and transformed with oncogenic Ras to test whether ROS can enhance Ras induced-transformation. The tumor suppressor gene p53 is known to be a redox sensitive protein. To study whether p53 is affected by ROS and whether this can lead to enhanced transformation susceptibility by Ras, primary MEFs from p53 and Prdx1 deficient mice will be transformed after chronic stress treatment with H202 and oncogenic Ras.